Bacteriophage phi29 of Bacillus subtilis is a small, well characterized virus containing a double-stranded DNA of 18 Kbp. Its entire genome has been sequenced and each of twenty-three genes is marked with suppressor-sensitive (sus) or temperature sensitive (ts) mutants. The products of most of the genes have been identified, and structural proteins are assembled in a single morphogenetic pathway. We have described a completely defined in vitro assembly system in which the phi29 DNA with covalently bound gp3 is packaged into a prohead with the aid of the single protein gp16 (PNAS, 83, 3505, 1986) and with an efficiency equivalent to in vivo assembly. The present proposal is centered on an analysis of the topology of packaged DNA-gp3, and the structure and function of the DNA packaging protein gp16, a DNA-dependent ATPase that topologically modifies phi29 DNA- gp3 as a prerequisite for packaging. We will study the intravirion topology of DNA-gp3 by ion etching, which progressively erodes virion components from the outside to the inside while preserving the overall structure. By use of this method and virions assembled in vitro, we will determine 1) the location of the left and right ends of the genome in the capsid; 2) the location of gp3; and 3) the presence of sharp (180 degrees) bends in the parallel DNA strands. We will study the binding of the DNA-gp3 packaging protein gp16 to DNA-gp3 that induces topological modification of the DNA during in vitro assembly. We will study the stoichiometry and specificity of binding of gp16 to DNA-gp3. Interaction of gp16 with specific segments of DNA will be studied by footprinting or by use of methylation. Negative superhelical density in DNA/gp3-gp16 complexes will be measured by the use of psoralen photoaffinity probes and the rate of photobinding. We will seek evidence for formation of nucleosome-like structures with an altered DNA helical pitch by the use of nuclease digestion experiments. The molecular structure of gp16 will be studied. We will attempt to identify the functional domains of gp16 involved in prohead, DNA-gp3, and ATP interactions. We will perturb local structures of the protein by cloning altered DNA and characterizing genetically altered proteins for function in DNA binding and topological modification. We will attempt to produce 2-D and 3-D crystals of gp16; 2-D crystals will be analyzed by Fourier filtering of electron micrographs and 3-D crystals will be analyzed by X-ray diffraction.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE008515-04
Application #
3222257
Study Section
Special Emphasis Panel (SRC)
Project Start
1987-09-01
Project End
1992-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Dentistry
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Wichitwechkarn, J; Johnson, D; Anderson, D (1992) Mutant prohead RNAs in the in vitro packaging of bacteriophage phi 29 DNA-gp3. J Mol Biol 223:991-8
Guo, P X; Rajagopal, B S; Anderson, D et al. (1991) sRNA of phage phi 29 of Bacillus subtilis mediates DNA packaging of phi 29 proheads assembled in Escherichia coli. Virology 185:395-400
Grimes, S; Anderson, D (1990) RNA dependence of the bacteriophage phi 29 DNA packaging ATPase. J Mol Biol 215:559-66
Anderson, D; Bodley, J W (1990) Role of RNA in bacteriophage phi 29 DNA packaging. J Struct Biol 104:70-4
Grimes, S; Anderson, D (1989) In vitro packaging of bacteriophage phi 29 DNA restriction fragments and the role of the terminal protein gp3. J Mol Biol 209:91-100
Wichitwechkarn, J; Bailey, S; Bodley, J W et al. (1989) Prohead RNA of bacteriophage phi 29: size, stoichiometry and biological activity. Nucleic Acids Res 17:3459-68